Genetic Modification of the

Soybean to Enhance the

b-Carotene Content through Seed-
Specific Expression
KELOMPOK 7

▰ Maulia Widi (1633010038)

▰ Angelica Apnia P. (1633010039)
▰ Cecilia Vienna E. (1633010070)
▰ Setiarini Merdekawati (1633010071)

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 INTRODUCTION

Carotenoids are also precursors of abscisic acid (ABA),

which regulates plant growth, seed dormancy, embryo
development and stress responses . In addition, some carotenoids,
including b-carotene, are precursors of vitamin A which is
essential for human health. Vitamin A deficiency (VAD) leads to
severe clinical symptoms associated with night blindness,
xerophthalmia and breakdown of the human immune system.

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 Soybean [Glycine max (L.) Merrill] is one of the world’s most
important crops as not only an important source of oil and protein, but also
secondary metabolites. Since transgenic soybean plants have been steadily
developed since the 1980s, the introduction of insect and herbicide resistance,
has greatly enhanced soybean production worldwide. Moreover, improvements
in the efficiency of soybean transformation techniques have been continuously
required for functional genomics research and crop improvement applications.

In our present study, we genetically manipulated the carotenoid

biosynthetic pathway in soybean plants using an efficient Agrobacterium-
mediated transformation method and the PAC recombinant gene. In this study
describe the effectiveness of using the PAC gene in soybean to produce high
levels of b-carotene when expressed in a seed-specific manner.
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 MATERIAL AND METHODS

Methods :
 Soybean Transformation
 Genomic DNA Analysis
 RNA Analysis
 Carotenoid Extraction and HPLC Analysis
 Tocopherol and Phytosterol Extraction and GC-
TOFMS Analysis
 Determination of the Antioxidant Activity
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 Vector Construction and
Agrobacterium Transformation

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Soybean Transformation

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Genomic
Genomic DNA Analysis
DNA
Analysis

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 RESULTS

• Korean Soybean Transformants Using an

Efficient Agrobacterium-mediated Method
To enhance the nutritional characteristics of soybean
plants through increased levels of b-carotene, the
carotenoid biosynthetic pathway was genetically
engineered to express the recombinant PAC gene
using two different promoters: b-conglycinin for
seedspecific expression and CaMV-35S for
constitutive over-expression.
Herbicide-resistance in transgenic soybean plants was
examined using a leaf painting assay in which the
surfaces of two trifoliate leaves were brushed with
PPT solution (100 mg/L). At one wee after this
herbicide application, healthy transgenic leaves were
discernible from the non-transgenic leaves as they did
not wilt and die (Fig. 2 h).
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• Inspection of Seed Color Change through the Introduction of the Recombinant PAC
Gene
Among the T1 seeds obtained, the seed color change in four b-PAC lines from yellow to orange
was determined by visual inspection (Fig. 3a). These seeds were preferentially selected as b-
PAC transgenic lines whilst none of 35S-PAC lines showed any seed color changes. The
maintenance of the orange seed color was determined for the T2 seed generation (Fig. 3b) and
this color intensity was found to be stronger in the T2 seeds from an analysis of their cross
sections, suggesting a higher accumulation of carotenoids by gene homozygosis as the progress
of generation (Fig. 3c). In contrast, none of the T2 seeds in any of the 35S-PAC transgenic lines
showed any changes in seed color, as found also in the non-transgenic and empty-vector
transgenic soybean seeds.

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• Integration and Expression of Transgenes in Soybean
Plants
The intactness of the introduced gene regions was
examined by further genomic PCR analysis and successful
introduction of a complete transgene was confirmed in
almost all of our putative transformants. Five out of 24 b-
PAC and 1/19 of the 35S-PAC lines had missing regions of
the integrated gene. A final total of 37 transgenic lines
(19 b-PAC and 18 35S-PAC) were thus obtained using
Agrobacterium-mediated transformation.
For analyze the expression level of the PAC transgene for
the different promoters is in the seeds, both PCR assays
revealed a much stronger expression of the PAC gene in
the b-PAC compared with the 35S-PAC transgenic lines,
indicating that authentic seed-specific expression is
driven by the b-conglycinin promoter (Fig. 5a). On the
other hand, the levels of PAC gene expression in leaves
were found to be higher in the 35S-PAC lines (Fig. 5b).

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• Carotenoid Composition in the b-PAC and 35S-
PAC
In the seeds, the b-PAC soybean lines produced very
high levels of total carotenoids (an average of 110.7
g/g), which were 45-fold higher than these levels in the
35S-PAC lines (an average of 2.5 g/g) and the
negative control non-transgenic (2.4 g/g) and empty
vector-transgenic (1.6 g/g) soybean plants.
Hence, the use of the seed-specific bconglycinin
promoter successfully yielded a high accumulation of
b-carotenoids in soybean seeds.
The total carotenoid levels in the leaves was slightly
higher in the b-PAC (an average of 1,008 g/g)
compared with the 35S-PAC (an average of 875 g/g)
transgenic plants and negative controls
(882 and 951 g/g, respectively).
These data suggested that exogenous PAC gene
expression in the soybean leaves does not affect the
total carotenoid level or the b- to a-carotenoid ratio
regardless of the specificity of the promoter used 13
• Tocopherol and Phytosterol Compositions
in the b-PAC and 35S-PAC Transgenic
Soybean Seeds
The mean total tocopherol levels, including a-, b-,
c- and d-tocopherols, were similar in the b-PAC
seeds but were 1.5-fold higher in the 35S-PAC
seed, compared with the non-transgenic control
(Fig. 8a). In addition, quantification of phytosterols
including aamyrin, b-amyrin, b-sitosterol,
campesterol and stigmasterol showed 1.3 times
higher of total level due to the increase of
bsitosterol in b-PAC than 35S-PAC and non-
transgenic control
(Fig. 8b).
These results indicate that the expression of
carotenoid
biosynthetic genes in soybean plants differently
regulates the levels
of two lipophilic products synthesized via pathways
linked to the
isoprenoids, including the carotenoids. 14
• Through this DPPH assay, the
radical scavenging effect
using the methanol extracts of
b-PAC and 35SPAC transgenic
soybean seeds were measured
(Table 1).

As results, the radical scavenging

activity of 35S-PAC transgenic
soybean seeds showed the similar
level to two control plants of NT and
EV.
While, All b-PAC transgenic soybean
seeds showed higher radical
scavenging activity than others. It
strongly suggested that the
accumulation of carotenoids
including b-carotene might be
responsible for the antioxidant
activity through radical scavenging.
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 DISCUSION

Value improvements to the commercially important soybean

plant using biotechnology have increased with the development of
better transformation technologies. Since the CN method was
applied to 28 diverse cultivars and/or genotypes of the soybean,
improvements to this protocol have been suggested using half-seed
explants of the Williams82 soybean cultivars (4.5%) which have
been reported to increase the efficiency of transformation

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Figure 4. Determination of insertion events of b-PAC and 35S-PAC transgenes.
(a) Genomic Southern blot analysis was performed with genomic DNAs from each
leaf tissues and a PAC probe. The DNA molecular size markers are indicated on the
left. (b) Quantitative real-time PCR analysis was carried out with the same genomic
DNAs and a Bar primer set. Con, transgenic plant that its single Bar gene insertion
was already confirmed NT, non-transgenic plants; EV, empty vector-transgenic
plants. 17
 Our analysis of the soybean in this study has found that
the PAC gene can successfully function in both
monocotyledons and dicotyledonous plants when expressed
under the control of seed-specific promoters. The efficiency in
this regard was found to be quite high in our current
experiments with the transgenic soybean and showed a 175-
fold higher level of b-carotene formation.

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 Of particular note, we here report one of the
highest levels of b-carotene production in
soybean seeds (112 g/g DW in b-PAC 16 line)
when compared with previously reported
results in transgenic plants.

This current generation of high b-carotene

soybean crops has the potential to help alleviate
VAD in humans and to provide an important
nutrition balance as a biofortified food for
livestock with provitamin A and antioxidant
activity.

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Thank You!

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